Echinacea, Hypericum and Prunella have been found to have appreciable anti-inflammatory activity. This activity may result from 1) an inhibition of inflammatory factors and/or, 2) an enhancement of anti- inflammatory cytokines. The health benefits of Echinacea, Hypericum, and Prunella extracts may directly result from a reduction in inflammation and an associated reduction in symptoms, and these potential benefits may extend to diseases with an inflammatory component. This Project will focus on understanding the balance between the anti- and pro-inflammatory activities of these supplements, both in cell and animal models, and on probing their mechanisms of action in cell systems. The overarching hypothesis under investigation is that the anti-inflammatory activity of fractions prepared from Echinacea, Prunella, or Hypericum is due to interacting constituents that shift the complex balance of immune-modulators toward an anti-inflammatory profile by modulating key signal transduction pathways and subsequent gene expression. Three Specific Aims are proposed: 1) Assessing the effects of Echinacea, Prunella, and Hypericum perforatum accessions, fractions and compounds on pro-inflammatory / anti-inflammatory cytokine and chemokine balance, in cultured cell populations with and without immune stimulation (primary lymphocytes and monocyte/macrophages, and the following cell lines: the RAW264.7 macrophage cell line, the A549 human bronchial epithelial cell line, and the MODE-K murine colonic epithelial cell line). 2) Assessing the role of Echinacea, Hypericum and Prunella in minimizing immunopathology and inflammation in established animal models of respiratory viral infection that result in acute pulmonary inflammation (influenza) and inflammatory disease (dextran sulfate sodium [DSS]-induced colitis model of inflammatory bowel disease). 3) Evaluating the effects of fractions and constituents of Echinacea and H. perforatum accessions on cellular signaling pathways that regulate in the severity of inflammation. We will initially use microarraysto identify the key interacting pathways that are altered by constituents and fractions of Echinacea and Hypericum (Aim 3.a). We will then use western blots, phosphorylation analysis, immune complex kinase assays, and gel shift technologies to further delineate the underlying mechanisms that regulate the activation of the key pathways (Aim 3.b). The proposed studies will further our understanding of Echinacea, Hypericum and Prunella constituents that contribute to the anti-inflammatory activity of these genera and determine if these genera, may be of value in treating inflammatory processes in the lung and colon. Furthermore, the proposed studies will assess the cellular mechanisms underlying these benefits, focusing on signaling pathways that have been shown to be central in regulating pro- and anti-inflammatory processes.